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GROWTH, DIFFERENTIATION, AND APOPTOSIS
1Departments of Pharmacology, 2Biomedical Sciences, and 3Cell Biology and Neuroscience, College of Medicine, University of South Alabama, Mobile, Alabama; and 4Department of Chemical Engineering, University of California at Berkeley, Berkeley, California
Submitted 13 August 2007 ; accepted in final form 10 December 2007
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ABSTRACT |
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angiotensin II; mitochondrial permeability transition pore; NADPH oxidase
Interestingly, the majority of research investigating the role of mtDNA damage in disease has focused on the gradual accumulation of mutagenic oxidative base modifications in these pathologies. Indeed, several specific mtDNA point mutations and deletions have been linked to various types of cancers (19), diabetes (7), neurodegenerative disorders (33), and neuromuscular disorders (21). Furthermore, transgenic mice that accumulate excessive mtDNA damage have been shown to develop the metabolic syndrome (35), dilated cardiomyopathy and heart failure (17, 37), and die prematurely (15, 17, 34).
However, it is becoming increasingly clear that not all oxidative damage to mtDNA results in nucleotide substitution or mutation. Rather, single strand breaks as well as abasic sites are common forms of oxidative mtDNA damage. Recently, it has been estimated that, under normal physiological conditions, 
9,000 abasic sites form each day (38), any one of which, according to our hypothesis, can be cytotoxic and result in acute cell death. However, despite an association between the many diseases linked to oxidative stress and acute cell death, the role of single strand breaks and abasic sites in this process has received little attention.
We have previously suggested that oxidative mtDNA damage plays a key role in the mechanism of apoptosis induced by angiotensin II (10). Moreover, Rachek et al. (23) have demonstrated that mtDNA repair enhances survival in cells subjected to oxidative stress. While it is logical to conclude that the gradual accumulation of mutagenic lesions can cause tissue damage, we propose that a small number of single strand breaks and abasic sites within the mitochondrial genome can rapidly induce apoptosis long before mutations can accumulate. However, the mechanism by which these acute lesions can cause apoptosis and tissue dysfunction remains unclear.
Because postmitotic tissues, as well as tissues with high-energy requirements, contain more mitochondria and are thought to be especially prone to the damaging effects of oxidative species and mtDNA damage (2), the mechanism underlying the effects of these cytotoxic lesions was studied in cultured neonatal cardiomyocytes subjected to angiotensin II. Notably, angiotensin II is known to increase reactive oxygen species and promote apoptosis in cardiac myocytes (8, 10, 29). Moreover, angiotensin II levels increase during heart failure, one of the many mtDNA damage-linked diseases in which acute cell death is thought to play a role. The present findings uncover a novel mechanism that links oxidative mtDNA damage to mitochondrial dysfunction and apoptosis independent of mutations.
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MATERIALS AND METHODS |
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Lentiviral vector generation and overexpression of human 8-oxoguanine DNA glycosylase. cDNA encoding either nuclear or mitochondrially targeted human 8-oxoguanine DNA glycosylase was inserted into a lentiviral vector for delivery to cells, with expression driven from a human cytomegalovirus promoter (as described previously for neonatal cardiomyocytes, 25) using standard recombinant DNA methods. The vector was packaged by transient transfection of the vector plasmid and three helper plasmids (encoding the viral Gag-Pol, Rev and the envelope protein VSVG) into 293 human embryonic kidney cells (HEK293) using the calcium phosphate method. Virus secreted into the cell culture media was collected and concentrated by ultracentrifugation to a typical titer of 109 transducing units per milliliter, as assessed by quantitative PCR of HEK293 cell genomic DNA following infection. Forty eight hours before analysis, virus was incubated with the cardiomyocyte cultures at a multiplicity of infection of 5:1. Control cells were incubated with either empty lentiviral vector or lentiviral vector containing cDNA for green fluorescent protein.
Annexin V and propidium iodide procedures. To assess the number of apoptotic cells in control and human 8-oxoguanine overexpressing cells, the TACS Annexin V-FITC kit was used. After 4 h of exposure to control medium or medium supplemented with angiotensin II in the presence or absence of cyclosporin A (200 nM), the culture medium was removed from the cells. The cells were first rinsed with cold 1x PBS buffer and then placed in 100 µl of annexin V incubation reagent consisting of 10 µl of 10x binding buffer, 10 µl of propidium iodide, 1 µl of annexin V conjugate, and 79 µl of distilled water. After a 15-min incubation in the dark with the annexin V reagent, the cells were washed for 2 min with an excess of 1x binding buffer [100 mM HEPES (pH 7.4), 1.5 M NaCl, 50 mM KCl, 10 mM MgCl2, and 18 mM CaCl2]. Annexin V and propidium iodide staining were observed by fluorescence microscopy using an Olympus IX 70 inverted microscope. Five separate fields in microscope were examined for bright green (apoptotic) and red (necrotic) nuclei.
Western blot analysis. Cellular content of ND5, Ogg1, succinate dehydrogenase, inducible NOS (iNOS), β-actin, cytochrome c oxidase (cox3), and cytochrome c were determined by Western blot analysis. The cells were washed twice in buffer containing 250 mmol/l sucrose, 10 mmol/l triethanolamine, pH 7.6, at room temperature. Cells were then lysed in ice-cold isolation buffer (pH 7.25 at 4°C) with the following composition: 150 mmol/l mannitol, 2 mmol/l sucrose, 5 mmol/l HEPES, 1 mmol/l EDTA, and protein protease inhibitors (a 1:100 dilution of protease inhibitor cocktail set III, 1% phenylmethysulfonyl fluoride, and 100 mmol/l sodium orthovanadate). The homogenate was centrifuged (Micromax RF, Thermo) at 800 g for 6 min at 4°C, and the supernatant was retrieved and centrifuged again at 12,000 g for 15 min. The pellet was defined as the mitochondrial fraction and used to determine ND5, Ogg1, cox3, and succinate dehydrogenase content. The supernatant was defined as the cytosolic fraction and used to determine iNOS, β-actin, and cytochrome c content. Protein concentration was determined by the Lowry method using bovine serum albumin as a standard. During Western blot analysis, the samples were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred to nitrocellulose membranes, where they were blocked. After incubation with the appropriate antibody, the membranes were washed and then incubated with a secondary antibody. The Western blots were detected by the enhanced chemiluminescence reaction.
Quantitative real time PCR method of measuring levels of ND5 mRNA. Total RNA was isolated from cells using TRIzol LS Reagent (Invitrogen) according to the protocol. Quantitative real time PCR was then performed using the iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad, Hercules, CA) according to the protocol. Primer sequences for ND5 were 5'-acgcctagcattaggaagca-3 (forward) and 5'-ttctagggcaatggcaaatc-3' (reverse), and primer sequences used for β-actin were 5'-gtgacgttgacatccgtaaa-3 (forward) and 5'-ctcaggaggagcaatgatct-3' (reverse).
Detection of mitochondrial DNA damage using quantitative Southern blots.
Isolated neonatal cardiomyocytes were exposed to 1 nM angiotensin for 20 min, 40 min, or 1 h. Forty eight hours before exposure to 1 nM angiotensin II, some cardiomyocytes were transfected with either human 8-oxoguanine glycosylase or green fluorescent protein. After the cells were lysed, high-molecular-weight DNA was phenol extracted, precipitated with ammonium acetate, and then exposed to 2 volumes of cold ethanol. The DNA samples were resuspended in water and then digested with the restriction endonuclease BamHI (10 U/µg DNA) at the same time it was treated with DNAse-free RNase (1.0 /ml) for 12–16 h at 37°C. After digestion, the samples were precipitated with ammonium acetate and resuspended in Tris-EDTA buffer (10 mM Tris and 1 mM EDTA; pH 8.0). The DNA was precisely quantified using a Hoefer TKO 100 Mini-Fluorometer and a TKO standards kit (Hoefer Scientific Instruments; San Francisco, CA). Samples containing 5 µg DNA were heated at 65°C for 20 min and then cooled at room temperature for an additional 20 min. After NaOH was added to a final concentration of 0.1 N, samples were incubated for 15 min at 37°C. This produced single-strand breaks at any abasic site in the DNA. Samples were then combined with 5 µl of loading dye, loaded onto a 0.6% alkaline agarose gel, and electrophoresed at 30 V (1.5 V/cm gel length) for 16 h in an alkaline buffer consisting of 23 mM NaOH and 1 mM EDTA. The gels were stained with ethidium bromide to confirm equal loading. After the gels were washed, DNA was transferred to a Zeta-Probe GT nylon membrane (Bio-Rad). The membranes were cross-linked and hybridized with a 32P-labeled rat mitochondria DNA-specific PCR-generated probe. Hybridization and subsequent washes were performed according to the manufacturer's recommendations. DNA damage was assessed as the following: break frequency = –ln (band intensity after 1 nM angiotensin II)/(band intensity of control).
Measurement of oxygen consumption. Neonatal cardiomyocytes were incubated with angiotensin II (1 nM) for 0 (control), 60, 90, 120, 150, 180, or 240 min. Approximately 1 x 107 cells were then resuspended in control medium (1 ml) lacking angiotensin II and analyzed in a gas-tight vessel maintained at 37°C, equipped with a Clark-type oxygen electrode connected to a chart recorder, and calibrated. Uncoupled respiration was determined by adding carbonyl cyanide m-chlorophenylhydrazone (CCCP). To further evaluate flux through the electron transport chain, cells were permeabilized with digitonin (60 µg/mg protein) and resuspended in respiration buffer (1 ml) before analysis. Cells were then exposed to glutamate (10 mM) + malate (10 mM), and the response to ADP (0.5 mM) was monitored.
Detection of superoxide using dihydroethidium. Neonatal cardiomyocytes were incubated with dihydroethidium (DHE, 5 µM), which is oxidized to fluorescent ethidium in the presence of superoxide. After 1 h, the dye-containing medium was removed and replaced with fresh medium. Cells were then incubated for an additional 30 min before analysis. Changes in DHE fluorescence were monitored by confocal microscopy at 15-min intervals with at least 10 cells monitored per sample. There were no changes in fluorescence in cells not incubated with angiotensin II.
Detection of mitochondrial membrane potential using DiOC6(3). Neonatal cardiomyocytes were incubated with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3), 5 nM], which is taken up into the mitochondria of healthy cells where it fluoresces. After 1 h, the dye-containing medium was removed and replaced with fresh medium. Cells were then incubated for an additional 30 min before analysis. Changes in DiOC6(3) fluorescence were monitored by confocal microscopy at 15-min intervals, with at least 10 cells monitored per sample. To ensure mitochondrial-specific staining, DiOC6(3) was colocalized with MitoTracker Red.
Detection of nitric oxide using DAF-FM diacetate. Neonatal cardiomyocytes were incubated with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM, 5 µM), a nitric oxide-sensitive dye. After 30 min, the dye-containing medium was removed and replaced with fresh medium. Cells were then incubated for an additional 30 min before analysis. Changes in fluorescence were monitored utilizing a Nikon Eclipse TE2000-U inverted fluorescence microscope at 15-min intervals with at least 10 cells monitored per sample. There were no changes in fluorescence in cells not incubated with angiotensin II.
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RESULTS |
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80–100 min after the addition of the inhibitor (Fig. 1C). Initiation of the second oxidative phase coincided with the loss of mitochondrial membrane potential, suggesting that the second phase, but not the first phase, was responsible for the decrease in mitochondrial membrane potential (Fig. 1C). NAD(P)H oxidase and the mitochondrial electron transport chain represent the major sources of superoxide in the myocyte (18). To determine whether the mitochondrial electron transport chain was the source of the second oxidative phase, cells were loaded with the mitochondrial-specific, superoxide-dependent dye MitoSox. In support of a mitochondrial origin, angiotensin II-induced increases in MitoSox fluorescence coincided with phase 2 oxidation (Fig. 1, A and C). Conversely, inhibition of the mitochondrial electron transport chain blocked phase 2 oxidation (data not shown). Together, these findings suggest that treatment with angiotensin II yields two overlapping phases of superoxide generation that can be resolved into distinct peaks via an NAD(P)H oxidase inhibitor.
Grishko et al. (10) had previously introduced the concept that both oxidative and nitrosative stress contribute to angiotensin II toxicity. To begin addressing whether nitric oxide and nitrosative stress could contribute to phase 2 oxidation, the time course of iNOS activation was examined. As seen in Fig. 2, iNOS is upregulated within 30 min of angiotensin II treatment. Although the levels of iNOS continued to increase between 30 and 60 min, Figs. 1 and 2 reveal that the generation of both superoxide [from NAD(P)H oxidase] and nitric oxide (from iNOS) preceded the onset of phase 2 oxidation.
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30% following exposure to angiotensin II (Fig. 5B). The angiotensin II-mediated decline in ND5 protein levels was blocked by the pretreatment of the cells with the NAD(P)H oxidase inhibitor apocynin but not when the addition of the inhibitor was delayed by 40 min (Fig. 5A).
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To evaluate the effect of mtDNA damage on the cardiomyocytes, cells were infected with a high titer lentiviral vector containing cDNA encoding for either a mitochondrially or nuclear targeted DNA repair enzyme human 8-oxoguanine DNA glycosylase. Forty eight hours following transduction, the levels of 8-oxoguanine glycosylase in the mitochondria were significantly elevated, indicating successful overexpression of the mitochondrial-targeted enzyme (Fig. 7). As observed in Fig. 4, these cells exhibited a significant reduction in the frequency of angiotensin II-induced mtDNA strand breaks (from 0.5 to 0.2 lesions/10 kbase). Importantly, this improvement in mtDNA was associated with an elevation in ND5 levels (Fig. 8), elimination of phase 2 oxidation despite the presence of a prominent phase 1 oxidative peak (Fig. 9A), and prevention of the decline in the mitochondrial membrane potential (Fig. 9B).
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DISCUSSION |
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The observation that overexpression of 8-oxoguanine glycosylase in the mitochondria, which reduces the number of abasic sites and strand breaks, also provides important insight into the cytoprotective mechanism of the repair enzyme. Through a process of base excision repair, 8-oxoguanine glycosylase recognizes and removes 8-oxoguanine, a major oxidative base lesion in mtDNA (27). More importantly, the repair enzyme also exhibits AP lyase activity, which cleaves the DNA phosphate backbone 3' of the lesion to help repair abasic sites and some DNA strand breaks. It has been documented that peroxynitrite can directly initiate DNA strand breaks and abasic sites (4, 20, 32), both which interfere with DNA transcription. Moreover, the reaction of the oxidant directly with DNA bases and deoxyribose causes further DNA damage (4, 20, 32). Absent this repair, the immediate effect of this damage would be a decline in the expression of mitochondrially encoded proteins, resulting in the slowing of electron transport chain flux and promotion of mitochondrial superoxide generation.
Mitochondrial polymerase DNA polymerase gamma will frequently miscode adenine opposite 8-oxoguanine during replication (27). Although this reaction would lead to a mutation, several lines of evidence suggest that it is not a cause of pathology in the angiotensin II-treated cell. First, a variety of mutagenic base modifications are alkali labile, therefore, NaOH treatment should provide a substantially distinct pattern of angiotensin II-induced mtDNA damage compared with cells not treated with NaOH. Yet, as demonstrated by Grishko et al. (10), the LM-PCR-based pattern of angiotensin II-induced mtDNA damage was similar whether or not the mtDNA was first treated with NaOH. Second, the formation of mutations is a slow process compared with the relatively rapid response to DNA strand breaks and abasic sites. Indeed, the effects of a mutation would only be noted after sufficient mtDNA damage has occurred, the damaged DNA is replicated [a process that requires a minimum of 60 min to complete, (9)], the mutated DNA is transcribed, and the modified protein has time to modulate mitochondrial function. By contrast, Fig. 5 shows that ND5 mRNA levels decrease within 30 min of the initial sign of mtDNA damage, a time course inconsistent with gradual accumulation of mutations. Third, no new protein bands were detected by the ND5 antibody following angiotensin II exposure (data not shown).
Unlike mutagenic lesions, abasic sites and single strand breaks create a block to replication and transcription (3, 28). Since transcription of most of the mitochondrially encoded polypeptides is driven by a single promoter (30), a single abasic site or single strand break could prevent the expression of all polypeptides located downstream from the damage, resulting in an immediate and dramatic decrease in the expression of mitochondrial proteins that make up the electron transport chain. One of these proteins is ND5, which combines with nuclear encoded and mitochondrially encoded polypeptides to form complex 1 of the electron transport chain (6). As seen in Figs. 4 and 5, the progressive reduction in ND5 mRNA and protein levels rapidly followed the onset of mtDNA damage.
We propose that oxidation of mtDNA is a key irreversible step in the signaling pathway. In support of this notion, Fig. 9 reveals that either overexpression of 8-oxoguanine glycosylase in the mitochondria or treatment with a NOS inhibitor, conditions that minimize mtDNA damage, abolishes phase 2 oxidation. Phase 2 oxidation is also abolished by pretreatment with an NAD(P)H oxidase inhibitor. In all of these conditions in which mtDNA damage was limited, the downstream expression of the mitochondrial proteins was robust and electron transport was sufficient to prevent the diversion of electrons to oxygen. However, prevention of further mtDNA damage after the initial insult by delayed addition of either a NOS inhibitor or an NAD(P)H oxidase inhibitor did not rescue the decline in ND5 levels or the appearance of phase 2 oxidation. Therefore, only the initial mtDNA damage is necessary to trigger the entire apoptotic pathway.
A key element of this novel death cascade is the existence of two distinct sources of superoxide that uniquely link the cytosol and mitochondria. Cytosolic NAD(P)H oxidase participates in the death cascade by providing one source of superoxide (phase 1 oxidation). As seen in Fig. 1A, superoxide levels increase rapidly following exposure to angiotensin II. Inhibition of phase 1 superoxide generation prevents mitochondrial DNA damage and angiotensin II-mediated apoptosis (10). Before cell death, however, superoxide combines with nitric oxide to form peroxynitrite. Although peroxynitrite is known to be the oxidizing agent responsible for angiotensin II-induced mtDNA damage (10), it is not certain whether peroxynitrite is formed in the cytosol, as proposed in Fig. 10. Since mitochondria contain their own NOS (mtNOS) (36), it is possible that superoxide generated in the cytosol could combine with locally produced nitric oxide in the mitochondria to induce mtDNA damage. Under these circumstances, exposure to L-NMMA would still be predicted to provide protection consistent with that observed in Fig. 3 by inhibiting mtNOS, as opposed to iNOS in the cytosol. However, the notion that cytosolic superoxide can enter the mitochondria, against a strong concentration gradient, is unlikely. Conversely, cytosolic peroxynitrite is known to readily enter the mitochondria. Thus it seems more logical to propose that peroxynitrite is forming in the cytosol. Indeed, as seen in Fig. 2, there is significant nitric oxide in the cytosol, which increases following exposure to angiotensin II. Therefore, there appears to be a sufficient stimulation of both superoxide and nitric oxide generation to facilitate the formation of peroxynitrite, the oxidant involved in mtDNA damage.
Phase 2 oxidation arises from the impairment of flux through the electron transport chain, as evidenced by decreased ND 5 levels, complex 1 activity, and oxygen consumption. Abolishing phase 2 oxidation irrespective of the mode of inhibition [through 8-oxoguanine glycosylase overexpression, NAD(P)H oxidase inhibition, and iNOS inhibition] invariably leads to cell survival. Indeed, disruption at an early stage in the pathway is equally effective in preventing death as the disruption of phase 2 oxidation using the antioxidant Tiron, suggesting that death occurs as the antioxidant defenses are overwhelmed. Evidence that angiotensin II increases MitoSox fluorescence and promotes the loss of aconitase activity suggests that the mitochondrial antioxidant defenses are overwhelmed by oxidants generated within the mitochondria. According to Halestrap et al. (11), one of the major consequences of mitochondrial oxidative stress is the oxidation of key residues on adenine nucleotide translocase, which facilitates the interaction of this translocase with cyclophilin D and promotes MPT pore formation. The observation that cyclosporin A blocks angiotensin II-mediated apoptosis (Fig. 9D) supports our view that phase 2 oxidation triggers the MPT. This conclusion is also borne out by the observation that angiotensin II stimulates the release of cytochrome c from the mitochondria and promotes the collapse of the mitochondrial membrane potential (Fig. 9, B and C), both characteristic features of the MPT.
The novel mechanism described in the present study has direct relevance to heart failure and diabetes, diseases in which angiotensin II plays a central role (8). However, the pathway appears to have wider implications. Druzhyna et al. (23) found that the oxidant menadione is also capable of initiating an acute apoptotic cascade, although the steps involved in that death pathway were not determined. Since oxidative stress is a key feature of cancer, certain neurodegenerative diseases and aging, the novel death cascade initiated by mtDNA damage may be important in a variety of pathological conditions.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Bohr VA. Repair of oxidative DNA damage in nuclear and mitochondrial DNA, and some changes with aging in mammalian cells. Free Radic Biol Med 32: 804–812, 2002.[CrossRef][Web of Science][Medline]
3. Boiteux S, Guillet M. Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae. DNA Repair 3: 1–12, 2004.[CrossRef][Medline]
4. Burney S, Caulfield JL, Niles JC, Wishnok JS, Tannenbaum SR. The chemistry of DNA damage from nitric oxide and peroxynitrite. Mutat Res 424: 37–49, 1999.[Web of Science][Medline]
5. Calabrese V, Lodi R, Tonon C, D'Agata V, Sapienza M, Scapagnini G, et al. Oxidative stress, mitochondrial dysfunction and cellular stress response in Friedreich's ataxia. J Neurol Sci 233: 145–162, 2005.[CrossRef][Web of Science][Medline]
6. Carroll J, Fearnley IM, Shannon RJ, Hirst J, Walker JE. Analysis of the subunit composition of complex I from bovine heart mitochondria. Mol Cell Proteomics 2: 117–26, 2003.
7. de Andrade PB, Rubi B, Frigerio F, van den Ouweland JM, Maassen JA, Maechler P. Diabetes-associated mitochondrial DNA mutation A3243G impairs cellular metabolic pathways necessary for beta cell function. Diabetologia 49: 1816–1826, 2006.[CrossRef][Web of Science][Medline]
8. Fiordaliso F, Li Baosheng Latini R, Sonnenblick EH, Anversa P, Leri A, Kajstura J. Myocyte death in streptozotocin-induced diabetes in rats is angiotensin II-dependent. Lab Invest 80: 513–527, 2000.[Web of Science][Medline]
9. Graves SW, Johnson AA, Johnson KA. Expression, purification, and initial kinetic characterization of the large subunit of the human mitochondrial DNA polymerase. Biochemistry 37: 6050–6058, 1998.[CrossRef][Web of Science][Medline]
10. Grishko V, Pastukh V, Solodushko V, Gillespie M, Azuma J, Schaffer SW. Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage. Am J Physiol Heart Circ Physiol 285: H2364–H2372, 2003.
11. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion–a target for cardioprotection. Cardiovasc Res 61: 372–385, 2004.
12. Halliwell B. Oxidative stress and cancer: have we moved forward? Biochem J 401: 1–11, 2007.[CrossRef][Web of Science][Medline]
13. Ide T, Tsutsui H, Kinugawa S, Suematsu N, Hayashidani S, Ichikawa K, Utsumi H, Machida Y, Egashira K, Takeshita A. Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium. Circ Res 86: 152–157, 2000.
14. Jones DP. Disruption of mitochondrial redox circuitry in oxidative stress. Chem Biol Interact 163: 38–53, 2006.[CrossRef][Web of Science][Medline]
15. Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, Hofer T, Seo AY, Sullivan R, Jobling WA, Morrow JD, Van Remmen H, Sedivy JM, Yamasoba T, Tanokura M, Weindruch R, Leeuwenburgh C, Prolla TA. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 309: 481–484, 2005.
16. Lankin ND, Jackson SP. Regulation of p53 in response to DNA damage. Oncogene 18: 7644–7655, 1999.[CrossRef][Web of Science][Medline]
17. Lewis W, Day BJ, Kohler JJ, Hosseini SH, Chan SS, Green EC, Haase CP, Keebaugh ES, Long R, Ludaway T, Russ R, Steltzer J, Tioleco N, Santoianni R, Copeland WC. Decreased mtDNA, oxidative stress, cardiomyopathy, and death from transgenic cardiac targeted human mutant polymerase gamma. Lab Invest 87: 326–335, 2007.[Web of Science][Medline]
18. Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, Melendez JA, Robinson JP. DPI induces mitochondrial superoxide-mediated apoptosis. Free Radic Biol Med 34: 465–477, 2003.[CrossRef][Web of Science][Medline]
19. Mandavilli BS, Santos JH, Van Houten B. Mitochondrial DNA repair and aging. Mutat Res 509: 127–151, 2002.[Web of Science][Medline]
20. Niles JC, Wishnok JS, Tannenbaum SR. Peroxynitrite-induced oxidation and nitration products of guanine and 8-oxoguanine: structures and mechanisms of product formation. Nitric Oxide 14: 109–121, 2006.[CrossRef][Web of Science][Medline]
21. Peng TI, Yu PR, Chen JY, Wang HL, Wu HY, Wei YH, Jou MJ. Visualizing common deletion of mitochondrial DNA-augmented mitochondrial reactive oxygen species generation and apoptosis upon oxidative stress. Biochim Biophys Acta 1762: 241–255, 2006.[Medline]
22. Qin F, Patel R, Yan C, Liu W. NADPH oxidase is involved in angiotensin II-induced apoptosis in H9C2 cardiac muscle cells: effects of apocynin. Free Radic Biol Med 40: 236–246, 2006.[CrossRef][Web of Science][Medline]
23. Rachek LI, Grishko VI, LeDoux SP, Wilson GL. Role of nitric oxide-induced mtDNA damage in mitochondrial dysfunction and apoptosis. Free Radic Biol Med 40: 754–762, 2006.[CrossRef][Web of Science][Medline]
24. Robertson RP, Harmon JS. Diabetes, glucose toxicity, and oxidative stress: a case of double jeopardy for the pancreatic islet beta cell. Free Radic Biol Med 41: 177–184, 2006.[CrossRef][Web of Science][Medline]
25. Robolledo MA, Krogstad P, Chen F, Shannon KM, Klitzner TS. Infection of human fetal cardiac myocytes by a human immunodeficiency virus-1-derived vector. Circ Res 83: 738–742, 1998.
26. Rosen P, Wiernsperger NF. Metformin delays the manifestation of diabetes and vascular dysfunction in Goto-Kakizaki rats by reduction of mitochondrial oxidative stress. Diabetes Metab Res Rev 22: 323–330, 2006.[CrossRef][Web of Science][Medline]
27. Russo MT, DeLuca G, Degan P, Bignami M. Different DNA repair strategies to combat the threat from 8-oxoguanine. Mutat Res 614: 69–76, 2007.[Web of Science][Medline]
28. Sanchez G and Mamet-Bratley MD. Transcription by T7 RNA polymerase of DNA containing abasic sites. Environ Mol Mutagen 23: 32–36, 1994.[Web of Science][Medline]
29. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res 91: 406–413, 2002.
30. Shoubridge EA. The ABCs of mitochondrial transcription. Nat Genet 31: 227–228, 2002.[CrossRef][Web of Science][Medline]
31. Stevnsner T, Thorslund T, de Souza-Pinto NC, Bohr VA. Mitochondrial repair of 8-oxoguanine and changes with aging. Exp Gerontol 37: 1189–1196, 2002.[CrossRef][Web of Science][Medline]
32. Szabo C, Ohshima H. DNA damage induced by peroxynitrite: subsequent biological effects. Nitric Oxide 1: 373–385, 1997.[CrossRef][Web of Science][Medline]
33. Szeto HH. Mitochondria-targeted peptide antioxidants: novel neuroprotective agents. AAPS J 8: E521–E531, 2006.[CrossRef][Web of Science][Medline]
34. Trifunovic A, Hansson A, Wredenberg A, Rovio AT, Dufour E, Khvorostov I, et al. Somatic mtDNA mutations cause aging phenotypes without affecting reactive oxygen species production. Proc Natl Acad Sci USA 102: 17993–17998, 2005.
35. Vartanian V, Lowell B, Minko IG, Wood TG, Ceci JD, George S, et al. The metabolic syndrome resulting from a knockout of the NEIL1 DNA glycosylase. Proc Natl Acad Sci USA 103: 1864–1869, 2005.[CrossRef][Web of Science]
36. Zenebe WJ, Nazarewicz RR, Parihar MS, Ghafourifar P. Hypoxia/reoxygenation of isolated rat heart mitochondria causes cytochrome c release and oxidative stress; evidence for involvement of mitochondrial nitric oxide synthase. J Mol Cell Cardiol 43: 411–419, 2007.[CrossRef][Web of Science][Medline]
37. Zhang D, Mott JL, Farrar P, Ryerse JS, Chang SW, Stevens M, et al. Mitochondrial DNA mutations activate the mitochondrial apoptotic pathway and cause dilated cardiomyopathy. Cardiovasc Res 57: 147–157, 2003.
38. Zhao H, Shen J, Deininger P, Hunt JD. Abasic sites and survival in resected patients with non-small cell lung cancer. Cancer Lett 246: 47–53, 2006.[Web of Science][Medline]
39. Zuckerbraun BS, Chin BY, Bilban M, de Costa d'Avila J, Rao J, Billiar TR, Otterbein LE. Carbon monoxide signals via inhibition of cytochrome c oxidase and generation of mitochondrial reactive oxygen species. FASEB J 21: 1099–1106, 2007.
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m. The results from the other subset of cells are not shown but they exhibit a separate pattern (no phase 2, no change in 
) were pretreated with NG-monomethyl-L-arginine (L-NMMA, 100 µM) to inhibit nitric oxide synthase (NOS) while others were left temporarily untreated (
). A: cells were then exposed to angiotensin II (1 nM), and changes in ethidium fluorescence were monitored using confocal microscopy. After 40 min apocynin (0.5 mM) was added to the medium to inhibit NAD(P)H oxidase, and monitoring was continued. B: L-NMMA-pretreated cells (
) fluorescence. C: L-NMMA-pretreated (
) cells, all of which overexpressed human 8-oxoguanine glycosylase. C: after 48 h, cells were exposed to angiotensin II (1 nM) for 4 h. After cell lysis, the cytosolic and mitochondrial fractions were isolated. Proteins from the mitochondrial and cytosolic fractions were separated by 15% SDS-PAGE. The proteins were transferred to nitrocellulose membranes, blocked, and then exposed to an antibody directed against cytochrome c, cytochrome c oxidase (cox3), and actin. After exposure to goat anti-mouse IgG, bands were detected by the enhanced chemiluminescence reaction. Shown is a representative gel based on n = 5. D: before angiotensin II exposure (1 nM, 4 h), some control cells were transduced with nuclear hOgg1. Fifteen minutes before angiotensin II treatment, some cells were additionally exposed to cyclosporin A (200 nM). The cells were then exposed to angiotensin II (1 nM for 4 h). After addition of angiotensin II, cells were incubated for 2 h with the Annexin V incubation reagent to identify apoptotic cells and propidium iodide to distinguish necrotic cells. The number of cells showing positive staining for Annexin V and negative staining for propidium iodide was determined by fluorescence microscopy. Shown is the percentage of apoptotic (Annexin positive, propidium iodide negative) cells. Values are means ± SE of 4–6 preparations. In A, all cells overexpressing hOgg1 were examined. Among the nontransduced cells containing normal levels of Ogg, only the subset of cells that exhibited declines in 